Serotonin transporter (SERT) plays an important role in the action and recycling of serotonin in the brain. SERT is a plasma membrane protein that binds serotonin in the extracellular space near a synapse, transports it across the membrane, and releases it to the cytoplasm. It is the target for drugs of abuse such as ecstasy (3.4-methylenedioxymethamphetamine, MDMA) and cocaine, as well as therapeutic drugs such as antidepressants like Prozac and imipramine. It is closely related to other neurotransmitter transporters such as those for dopamine, norepinephrine, GABA and glycine. As part of a wider interest in the structure and function of SERT, it has become apparent that the part of the transporter that faces the cytoplasm undergoes important conformational changes during transport. This intracellular domain consists of the NH2- and COOH-termini of the protein and five intracelluar loops. The current proposal seeks to understand the structure and function of these intracellular elements in three specific aims. The first of these is to scan the intracellular loops by cysteine mutagenesis to determine the boundaries between the loop and transmembrane domains. The second aim is to examine the possibility that intracellular loops interact specifically with each other to form the intracellular structure of the transporter. The third aim is to test the hypothesis that the translocation pathway for serotonin is formed, in part, by loops as well as transmembrane domains. It is expected that the results of these experiments will shed light on the structure and function of the related dopamine and norepinephrine transporters as well as of other members of the neurotransmitter transporter family.